PROJECT SUMMARY Clostridium difficile is a Gram-positive, rod-shaped, spore-forming, obligately anaerobic, toxigenic bacterium that is the leading cause of hospital acquired antibiotic-associated diarrhea worldwide. Along with two other antibiotic-resistant pathogenic bacteria, the CDC classifies C. difficile as an immediate public health threat that requires urgent and aggressive action. Accumulating evidence indicates that during infection C. difficile modifies its energy metabolism in response to changing nutrient conditions in the gut. Our results show that C. difficile possesses substantially greater metabolic versatility than previously appreciated, and is capable of rapidly shifting its metabolism to grow in the absence of several amino acids that until now were considered to be essential. Efficient growth on glucose in the absence of all major Stickland acceptor amino acids implicates an ability of C. difficile to readily adapt to a changing nutrient landscape in the infected host gut. Our studies further show that the Wood-Ljungdahl pathway, an energy-generating system in acetogens and certain other anaerobic bacteria and archaea, is dramatically up-regulated in response to the shift to growth on carbohydrates -- a nutrient source that host-derived glycans may provide under conditions of limited amino acid availability. Under these conditions, the central enzymes in the pathway, AcsB and CO dehydrogenase, attain physiologically relevant levels not previously observed in C. difficile. Our hypothesis is that the Wood- Ljungdahl pathway allows C. difficile to effectively adjust its energy metabolism and possible biosynthetic requirements in response to changing nutrient conditions in the gut. To test this hypothesis our specific aim is to carry out a detailed characterization of the properties of a C. difficile 630 acsB deletion mutant. The effects on growth, metabolite profiles, qRT-PCR analysis of various transcripts, phenotypic microarrays, the ability to produce toxin and effectively sporulate will be compared with the wild type under various growth-supportive nutrient conditions to provide a comprehensive characterization of the mutant. The results will elucidate how the Wood-Ljungdahl pathway is integrated in the metabolism of C. difficile, and define how the pathway acts to confer a metabolic advantage to this pathogen. The findings will direct future studies to evaluate the pathway as a potential target for prevention or treatment of C. difficile infection.